U.S. patent number 8,658,390 [Application Number 13/255,120] was granted by the patent office on 2014-02-25 for method for detecting muscle degenerative diseases, and method for determining therapeutic efficacy on the diseases.
This patent grant is currently assigned to National Center of Neurology and Psychiatry, Osaka Bioscience Institute, Taiho Pharmaceutical Co., Ltd.. The grantee listed for this patent is Kosuke Aritake, Shinya Kamauchi, Toshihiko Maruyama, Akinori Nakamura, Shin'ichi Takeda, Yoshihiro Urade. Invention is credited to Kosuke Aritake, Shinya Kamauchi, Toshihiko Maruyama, Akinori Nakamura, Shin'ichi Takeda, Yoshihiro Urade.
United States Patent |
8,658,390 |
Urade , et al. |
February 25, 2014 |
Method for detecting muscle degenerative diseases, and method for
determining therapeutic efficacy on the diseases
Abstract
Muscle degenerative diseases can be detected in the early stage
and the therapeutic efficacy of a therapeutic agent and/or a
therapy method for the diseases can be determined by measuring
11,15-dioxo-9.alpha.-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic
acid (referred to as "Tetranor-PGDM", hereinbelow) in a sample
isolated from a subject.
Inventors: |
Urade; Yoshihiro (Suita,
JP), Aritake; Kosuke (Suita, JP), Maruyama;
Toshihiko (Suita, JP), Kamauchi; Shinya (Suita,
JP), Takeda; Shin'ichi (Kodaira, JP),
Nakamura; Akinori (Kodaira, JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
Urade; Yoshihiro
Aritake; Kosuke
Maruyama; Toshihiko
Kamauchi; Shinya
Takeda; Shin'ichi
Nakamura; Akinori |
Suita
Suita
Suita
Suita
Kodaira
Kodaira |
N/A
N/A
N/A
N/A
N/A
N/A |
JP
JP
JP
JP
JP
JP |
|
|
Assignee: |
Osaka Bioscience Institute
(Suita-shi, Osaka, JP)
National Center of Neurology and Psychiatry (Kodaira-shi,
Tokyo, JP)
Taiho Pharmaceutical Co., Ltd. (Tokyo, JP)
|
Family
ID: |
42728314 |
Appl.
No.: |
13/255,120 |
Filed: |
March 8, 2010 |
PCT
Filed: |
March 08, 2010 |
PCT No.: |
PCT/JP2010/053762 |
371(c)(1),(2),(4) Date: |
September 07, 2011 |
PCT
Pub. No.: |
WO2010/104025 |
PCT
Pub. Date: |
September 16, 2010 |
Prior Publication Data
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|
|
|
Document
Identifier |
Publication Date |
|
US 20110318764 A1 |
Dec 29, 2011 |
|
Foreign Application Priority Data
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|
|
|
Mar 9, 2009 [JP] |
|
|
2009-055057 |
|
Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
G01N
33/6893 (20130101); G01N 2800/28 (20130101); G01N
30/7233 (20130101); G01N 2800/2878 (20130101); G01N
2030/8813 (20130101) |
Current International
Class: |
G01N
33/53 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
|
|
|
|
1911755 |
|
Apr 2008 |
|
EP |
|
2005-119984 |
|
May 2005 |
|
JP |
|
2010-02416 |
|
Jan 2010 |
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JP |
|
2009043015 |
|
Apr 2009 |
|
WO |
|
Other References
Takeshi Okinaga et al., "Induction of hematopoietic prostaglandin D
synthase in hyalinated necrotic muscle fibers: its implication in
grouped necrosis", Acta Neuropathol, 2002, vol. 104, No. 4, p.
377-384 Abstract. cited by applicant .
Wen-Liang Song et al., "Tetranor PGDM, an Abundant Urinary
Metabolite Reflects Biosynthesis of Prostaglandin D2 in Mice and
Humans", J Biol Chem, Jan. 11, 2008, vol. 283, No. 2, p. 1179-1188
Abstract, Figure 1. cited by applicant .
Cathy K. Ellis et al., "Metabolism of Prostaglandin D2 in the
Monkey", J Biol Chem, 1979, vol. 254, No. 10, p. 4152-4163; fig. 20
XIII, p. 4162, right column, lines 3 to 5. cited by applicant .
Ikuko Mohri et al., "Inhibition of Prostaglandin D Synthase
Suppresses Muscular Necrosis", Am J Pathol, May 2009, vol. 174, No.
5, p. 1735-1744; entire text. cited by applicant .
Shinya Kamauchi et al., "Dobutu Model ni Okeru Prostaglandin D2
Nyouchu Taishabutsu no Hendo", Journal of Japanese Biochemical
Society, Sep. 25, 2009, abstract CD Page.ROMBUN No. 2P-138; entire
text. cited by applicant .
M Kondo et al., 15-Deoxy-[Delta]<12, 14>-prostaglandin J2:
The endogenous electrophile that induces neuronal apoptosis,
Proceedings of the National Academy of Sciences of the United
States of America, vol. 99, No. 11, May 28, 2002, p. 7367-7372.
cited by applicant.
|
Primary Examiner: Ulm; John
Attorney, Agent or Firm: Rothwell, Figg, Ernst &
Manbeck, P.C.
Claims
The invention claimed is:
1. A method for detecting a muscle degenerative disease using a
urine sample, comprising obtaining a urine sample from a subject
suspected of suffering from a muscle degenerative disease;
measuring Tetranor-PGDM content in said urine sample; and comparing
the concentration of said Tetranor-PGDM with the concentration of
Tetranor-PGDM in urine from normal subjects, wherein a high
concentration of Tetranor-PGDM in said urine sample indicates the
possible presence of a muscle degenerative disease.
2. The method according to claim 1, wherein the Tetranor-PGDM is
measured by using high-performance liquid chromatography-tandem
mass spectrometry (HPLC-MS/MS), enzyme immunoassay (EIA),
radioimmunoassay (RIA), fluorescent immunoassay (FIA), ELISA, or an
enzymatic method.
3. The method according to claim 1, wherein the muscle degenerative
disease is progressive muscular dystrophy, congenital muscular
dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral
muscular dystrophy, myotonic muscular dystrophy, amyotrophic
lateral sclerosis, or myopathy.
4. A method for determining the efficacy of a therapeutic agent
and/or a therapeutic method for a muscle degenerative disease,
comprising obtaining a first urine sample from a subject prior to
undergoing therapy for a muscle degenerative disease; measuring
Tetranor-PGDM concentration in said first urine sample; obtaining a
second urine sample from a subject while undergoing therapy for a
muscle degenerative disease; measuring Tetranor-PGDM concentration
in said second urine sample; and comparing said Tetranor-PGDM
concentration in said first and second urine samples, wherein a
lower Tetranor-PGDM concentration in said second urine sample
indicates that the therapy is efficacious.
Description
CROSS REFERENCE TO RELATED APPLICATION
This application is a 35 U.S.C. 371 National Phase Entry
Application from PCT/JP2010/053762, filed Mar. 8, 2010, which
claims the benefit of Japanese Patent Application No. 2009-055057
filed on Mar. 9, 2009, the disclosure of which is incorporated
herein in its entirety by reference.
TECHNICAL FIELD
The present invention relates to a method for early detection of
muscle degenerative diseases, and a method for predicting and/or
determining a therapeutic agent and/or a therapeutic method.
BACKGROUND ART
Groups of diseases involving muscular disorder or myonecrosis are
called myopathy. Muscular dystrophy and amyotrophy are
representative examples of this class of disease. Muscular
dystrophy is a collective term used for hereditary diseases that
are characterized by gradual muscle weakening and atrophy.
Progressive muscular dystrophies affect the largest number of
patients, and cause hereditary, progressive muscle weakness.
Amyotrophy is a neurogenic disease caused by damages in motor
nerve.
The type of muscular dystrophy that affects the largest number of
patients is Duchenne muscular dystrophy, which is a sex-linked
recessive hereditary disease that develops only in males. The
disease affects 3 to 5 individuals per 100,000 people, and 1 in
2,000 to 3,000 newborn males. The disease generally develops at the
age of about 3 to 5 with defects in walking and standing, such as
running problems and frequent falls. The ability to walk is lost by
the age of around 10. These symptoms are followed by a rapid
progress of spinal column deformation and arthrogryposis, which in
many cases lead to respiratory failure, and, less often, heart
failure and pneumonia.
The tests used for the diagnosis of muscular dystrophy include a
blood test, a nerve conduction test, electromyography, a muscle
biopsy, and a DNA analysis. The nerve conduction test finds whether
mobility impairment or perception impairment stems from peripheral
neuropathy, or looks for a damaged site or the extent of damage.
The test measures the conduction rate of a stimulus in an
electrostimulated nerve. By nature, the test requires special
equipment, and, because an electrostimulation is directly applied
to the nerve, the test is somewhat demanding in the sense that it
involves shock, pain, and discomfort.
Electromyography finds whether mobility impairment originates in
muscle or nerve, or looks for a damaged site or the extent of
damage. The test requires special equipment, and involves pain from
the insertion of a needle into the muscle. Pain-free, surface
electromyography is available; however, the measurement must be
performed at test facilities.
Muscle biopsy requires collecting a muscle tissue, and is therefore
invasive and inconvenient. DNA analysis, necessary for the
diagnosis of Duchenne and Becker muscular dystrophies caused by a
mutation in the dystrophin gene, has not been applied to muscle
degenerative diseases, and lacks versatility.
The blood test generally looks for creatine kinase. Creatine kinase
is an enzyme predominantly present in the soluble fractions of
skeletal muscle and cardiac muscle, and leaks into the blood from
damaged cells. A damaged or dead skeletal muscle considerably
raises the blood creatine kinase levels, and such high levels of
blood creatine kinase can thus be used for the diagnosis of
muscular dystrophy. However, because the blood creatine kinase
levels can also increase in other diseases, a differential
diagnosis solely based on creatine kinase concentration is
difficult, and is made simultaneously with other tests.
The blood test that measures the blood creatine kinase is also
performed for other progressive muscular dystrophies, and for
diseases that involve muscle damage or death caused by nerve
defects. However, as above, high blood creatine kinase levels also
occur in diseases other than muscular disorders and myonecrosis,
and other markers for muscular disorders and myonecrosis are
needed.
Accordingly, there is a need for a method or a diagnosis kit that
enables an early and easy diagnosis of muscle degenerative diseases
such as muscular dystrophy.
11,15-Dioxo-9.alpha.-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic
acid (hereinafter, Tetranor-PGDM) is known as a metabolite of
prostaglandin D.sub.2 (hereinafter, PGD.sub.2), and there is a
report that the Tetranor-PGDM excreted into the urine increases
through inflammation reactions in humans and mice, and that
Tetranor-PGDM is a marker that reflects PGD.sub.2 production
(Non-Patent Literature 1).
There are also reports that the increased expression of
hematopoietic prostaglandin D synthetase (hereinafter, HPGDS) that
catalyzes PGD.sub.2 production occurs at the affected sites of
muscle degenerative diseases such as muscular dystrophy, and that
PGD.sub.2 is involved in the prevention and improvement of disease
progression (Patent Literature 1, Non-Patent Literature 2).
It is not known, however, that Tetranor-PGDM is detected in high
concentrations as an excretion in the urine of patients with muscle
degenerative diseases, and that the Tetranor-PGDM concentration
significantly decreases by the administration of an HPGDS
inhibitor.
CITATION LIST
Patent Literature
PTL 1: Japanese Unexamined Patent Publication No. 2005-119984
Non-Patent Literature
NPL 1: J. Biol. Chem., Vol. 283, No. 2, 1179-1188 (2008) NPL 2:
Acta Neuropathol, 104, 377-384 (2002)
SUMMARY OF INVENTION
Technical Problem
It is an object of the present invention to provide a method for
efficient diagnosis of a muscle degenerative disease through the
measurement of urine Tetranor-PGDM, and a method for determining
the therapeutic efficacy of a therapeutic agent and/or a
therapeutic method for such diseases.
Another object of the present invention is to provide a muscle
degenerative disease diagnosis kit that targets Tetranor-PGDM.
Solution to Problem
The present inventors conducted intensive studies to achieve the
foregoing objects, and completed the invention based on the
following findings. 1) A muscular dystrophy model animal had
elevated levels of the PGD.sub.2 metabolite Tetranor-PGDM in urine
compared to normal animals. 2) The administration of a known
PGD.sub.2 synthetase inhibitor to a muscular dystrophy model animal
lowered the amount of Tetranor-PGDM excreted into the urine.
The present invention provides a method for detecting a muscle
degenerative disease, a kit for the diagnostic measurement of a
muscle degenerative disease, and a kit for predicting and/or
determining the efficacy of a therapeutic agent and/or a
therapeutic method for muscle degenerative diseases, as
follows.
Item 1.
A method for detecting a muscle degenerative disease, the method
comprising the step of measuring a Tetranor-PGDM content in a
sample isolated from a subject.
Item 2.
A method for determining the efficacy of a therapeutic agent and/or
a therapeutic method for a muscle degenerative disease, the method
comprising the step of measuring a Tetranor-PGDM content in a
sample isolated from a muscle degenerative disease patient.
Item 3.
The method according to Item 1 or 2, wherein the sample is
urine.
Item 4.
The method according to any one of Items 1 to 3, wherein the
Tetranor-PGDM is measured by using high-performance liquid
chromatography-tandem mass spectrometry (HPLC-MS/MS), enzyme
immunoassay (EIA), radioimmunoassay (RIA), fluorescent immunoassay
(FIA), ELISA, or an enzymatic method.
Item 5.
The method according to Item 1 or 2, wherein the muscle
degenerative disease is progressive muscular dystrophy, congenital
muscular dystrophy, limb-girdle muscular dystrophy,
facioscapulohumeral muscular dystrophy, myotonic muscular
dystrophy, amyotrophic lateral sclerosis, or myopathy.
Item 6.
A diagnosis measurement kit for a muscle degenerative disease, the
kit comprising an antibody against Tetranor-PGDM.
Item 7.
A kit for predicting and/or determining the efficacy of a
therapeutic agent and/or a therapeutic method for a muscle
degenerative disease, the kit comprising an antibody against
Tetranor-PGDM.
Item 8.
The kit according to Item 6 or 7, comprising the antibodies against
Tetranor-PGDM, labeled Tetranor-PGDM, and, optionally, at least one
selected from the group consisting of an anti-immunoglobulin
antibody, a sample diluting solution, a diluting solution for the
antibody and the labeled Tetranor-PGDM, standard Tetranor-PGDM of a
known concentration, an EIA substrate, and an EIA stop
solution.
Advantageous Effects of Invention
The present invention enables an easy and early diagnosis of a
muscle degenerative disease through the measurement of
Tetranor-PGDM in a sample isolated from a subject, and can
effectively determine the therapeutic efficacy of a therapeutic
agent and/or a therapeutic method for such diseases.
The present invention also can be used as a diagnosis kit for an
easy diagnosis of muscle degenerative diseases, by using increased
urine Tetranor-PGDM as a marker.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a diagram showing changes in urine Tetranor-PGDM
concentration and forefoot grip strength in mdx mice administered
with an HPGDS inhibitor.
FIG. 2 is a diagram showing changes in urine Tetranor-PGDM
concentration after administering a solvent following about one
year of HPGDS inhibitor administration (left), and changes in
Tetranor-PGDM concentration in the urine of muscular dystrophy dog
(CXMDA that received the solvent for about one year before being
administered with the inhibitor (right).
DESCRIPTION OF EMBODIMENTS
The present invention enables the diagnosis of muscle degenerative
diseases using Tetranor-PGDM as an index, and can effectively
determine the therapeutic efficacy of therapeutic agents and/or
therapeutic methods for these diseases. Further, by using
Tetranor-PGDM as a marker, the invention can provide a diagnosis
kit for these diseases, or a kit for predicting and/or determining
the efficacy of therapeutic agents and/or therapeutic methods for
muscle degenerative diseases.
According to an embodiment of the present invention, a disease
involving muscular disorder or myonecrosis can be detected or
diagnosed by measuring the Tetranor-PGDM in a sample isolated from
a subject affected or potentially affected by muscle degenerative
disease. Specifically, the subject can be diagnosed with muscle
degenerative disease when the concentration or content of the
Tetranor-PGDM in a sample exceeds a predetermined value. The
predetermined value of the Tetranor-PGDM in a sample isolated from
a subject can be determined from the measured Tetranor-PGDM in
samples from a healthy individual and from a muscle degenerative
disease patient.
The method for determining the efficacy of therapeutic agents
and/or therapeutic methods compares the measured values of
Tetranor-PGDM in samples from a muscle degenerative disease patient
before and after the treatment/administration of a therapeutic
agent. The method determines that the treatment and the
administration of the therapeutic agent are effective when the
measured value of Tetranor-PGDM in the sample has lowered
significantly or marginally significantly after the
treatment/administration of the therapeutic agent. On the other
hand, the method determines that the therapeutic agent/therapeutic
method are ineffective when there is no significant or marginally
significant difference in the measured values of Tetranor-PGDM in
the sample before and after the treatment/administration of the
therapeutic agent.
According to another aspect of the present invention, a diagnosis
kit can be provided that uses antibodies for the detection of
Tetranor-PGDM in a sample.
As used herein, "subject" refers to mammals, including, for
example, humans, monkeys, bovines, horses, rats, mice, guinea pigs,
rabbits, dogs, cats, sheep, and goats. Preferably, the subject is a
human.
The Tetranor-PGDM measured by the method of the present invention
is found as a metabolite of PGD.sub.2 in urine. Tetranor-PGDM also
can be found in blood and feces. In the present invention, the
sample isolated from a subject is preferably urine, feces, blood,
blood plasma, or serum, more preferably urine.
As used herein, the term "measure" encompasses detection,
quantification, and semiquantification. As such, "measuring
Tetranor-PGDM" means both detecting Tetranor-PGDM in a sample, and
measuring the expression level. The term also encompasses
determining whether the expression level is at or above a
predetermined value, in other words, detecting expression when the
expression level is at or above a predetermined value.
Examples of the method that can be used to measure Tetranor-PGDM
include GC-MS, HPLC, high-performance liquid chromatography-tandem
mass spectrometry (HPLC-MS/MS), enzyme immunoassay (EIA),
radioimmunoassay (RIA), fluorescent immunoassay (FIA), ELISA, and
an enzyme method. Of these, high-performance liquid
chromatography-tandem mass spectrometry (HPLC-MS/MS) is preferred,
and, for ease of procedure, immunoassays using anti-Tetranor-PGDM
antibodies, specifically enzyme immunoassay (EIA), radioimmunoassay
(RIA), fluorescent immunoassay (FIA), and ELISA are preferred, and
enzyme immunoassay (EIA) and ELISA are particularly preferred.
Examples of the muscle degenerative disease include progressive
muscular dystrophy, congenital muscular dystrophy, limb-girdle
muscular dystrophy, facioscapulohumeral muscular dystrophy,
myotonic muscular dystrophy, amyotrophic lateral sclerosis,
myopathy, muscle strain, cardiomyopathy (myocardial infarction),
and diabetic peripheral vascular disease (vascular smooth muscle
disorder). Muscular dystrophies and amyotrophic lateral sclerosis,
such as progressive muscular dystrophy, congenital muscular
dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral
muscular dystrophy, and myotonic muscular dystrophy, are
preferred.
The therapeutic agent that can be used for the determination of
therapeutic efficacy for muscle degenerative diseases is not
particularly limited, and any therapeutic agent can be used,
including, for example, hematopoietic prostaglandin D synthetase
(HPGDS) inhibitors and prostaglandin D receptor antagonists, of
which hematopoietic prostaglandin D synthetase (HPGDS) inhibitors
are preferred.
It is preferable that the Tetranor-PGDM concentration in a sample
be measured by immunoassay, because it easily enables simultaneous
measurements of large sample numbers.
The anti-Tetranor-PGDM antibodies used for the immunoassay and the
kit may be, for example, polyclonal antibodies or monoclonal
antibodies.
With regard to antibody production, polyclonal antibodies and
monoclonal antibodies may be produced by administering
Tetranor-PGDM and immunizing an animal (rat, mouse, guinea pig,
rabbit, dog, cat, sheep, goat, etc.). Alternatively, polyclonal
antibodies and monoclonal antibodies may be obtained from the serum
collected from an animal (rat, mouse, guinea pig, rabbit, dog, cat,
sheep, goat, etc.) and treated by a known method after a
predetermined time period from the interval administration of the
animal with a suspension mixture of a suitable adjuvant and
Tetranor-PGDM bound to a suitable protein, for example, such as
bovine serum albumin (BSA), globulin, thyroglobulin, and
hemocyanin.
Specifically, monoclonal antibodies can be obtained from hybridomas
produced by fusing myeloma cells with monoclonal antibody-producing
cells obtained from spleen after immunizing an animal with an
immunogen, for which the Tetranor-PGDM used for the production of
polyclonal antibodies and optionally attached to a suitable protein
is used.
The hybridomas can be obtained as follows. The Tetranor-PGDM,
obtained as above either alone or as a complex with a protein, is
intraperitoneally, intravenously, or subcutaneously administered
with a complete Freund's adjuvant to a suitable animal (such as
mouse, rat, and rabbit) every 2 to 3 weeks in divided portions to
immunize the animal. The antibody-producing cells originating in
the spleen or other organs are then fused with tumor cells, such as
myeloma cells, that can proliferate in a test tube. The cells can
be fused by using polyethylene glycol according to the ordinary
method of Kohler and Milstein (Nature, vol. 256, 495 (1975)), or by
using Sendai virus.
The Tetranor-PGDM immunoassay is performed using the
anti-Tetranor-PGDM antibodies obtained as above. Preferably, the
immunoassay is performed by known competitive immunoassay methods
targeting the measured substance Tetranor-PGDM. Examples of such
methods include enzyme immunoassay (EIA), fluorescent immunoassay,
luminescent immunoassay, and radioimmunoassay (RIA), classified
according to the labeling substance. Of these, EIA is particularly
preferred.
Typically, labeled antigens are used for the competition method.
Examples of labeling substances include enzymes, fluorescent
substances, luminescent substances, and radioisotopes. The
conjugation between the labeling substance and antigens can be made
using known methods that form a covalent bond or a non-covalent
bond. Examples of such conjugation methods include a method that
forms a covalent bond using, for example, a condensing agent, and a
method that uses various crosslinkers (see, for example,
Tanpakushitsu Kakusan Kouso (PNE), Separate Volume 31, pp. 37 to 45
(1985)). The covalent binding method can be used to produce labeled
antigens by using the functional group present on the antigens, or
by binding a functional group such as a thiol group, an amino
group, a carboxyl group, and a hydroxyl group after introducing
these groups using an ordinary method. The non-covalent binding
method may be, for example, a physical adsorption method.
Preferably, Tetranor-PGDM is immunoassayed, for example, as
follows. Through a competition reaction between a predetermined
amount of labeled Tetranor-PGDM, anti-Tetranor-PGDM antibodies, and
a sample containing Tetranor-PGDM (particularly, a urine sample),
the Tetranor-PGDM in the sample is quantified from the amount of
the labeled antigens that have bound to the antibodies or did not
bind to the antibodies.
The labeled antigens bound to the antibodies can be isolated from
the unbound labeled antigens through addition of
anti-immunoglobulin antibodies and isolation of the precipitated
(labeled antigen)-(anti-Tetranor-PGDM
antibody)-(anti-immunoglobulin antibody) conjugates, followed by
the measurement of the labeling substance that has bound to the
conjugates or that did not bind to the conjugates. The method,
called a double antibody technique, also can be performed using a
method that uses a charcoal filter. The anti-immunoglobulin
antibody assay also can be performed by measuring the
anti-immunoglobulin antibodies that have bound to the solid phase,
or by measuring the labeling substance that has bound to the solid
phase or did not bind to the solid phase. The anti-immunoglobulin
antibodies may be bound to the solid phase by using known methods,
for example, such as a physical adsorption method, a chemical
binding method that uses a crosslinker or a covalent bond, and a
binding method that uses an avidin-biotin bond. The measurement of
the labeling substance should be selected according to the type of
labeling substance used.
The kit of the present invention includes anti-Tetranor-PGDM
antibodies. In a more preferred embodiment, the kit includes
labeled Tetranor-PGDM, and anti-Tetranor-PGDM antibodies. As
required, the kit may also include, for example,
anti-immunoglobulin antibodies that bind to the anti-Tetranor-PGDM
antibodies, a sample diluting solution, a diluting solution for the
antibodies and labeled Tetranor-PGDM, and standard Tetranor-PGDM of
a known concentration. For EIA, the kit may additionally include,
for example, a substrate and a stop solution.
The sample used for the measurement of Tetranor-PGDM in the present
invention may be specifically, for example, urine collected from
humans.
The efficacy determining method for muscle degenerative disease
patients compares the measured values of Tetranor-PGDM in a sample
(specifically, urine) before and after the administration of a
therapeutic agent.
The sample may be a pool of urine collected for a day, or a
collected sample may be directly used for the measurement. The
collected urine may be preserved at room temperature, preferably at
low temperature before use in the measurement.
The Tetranor-PGDM in a sample may be measured relative to the total
amount of the collected sample, or relative to a part of the
collected sample with consideration to correction by reference
substances such as creatinine.
For ease of procedure, the Tetranor-PGDM in a sample is preferably
measured relative to a part of the collected sample with
consideration to correction by creatinine.
The predetermined value used in the present invention is described
below.
The predetermined value used for the determination of therapeutic
efficacy for muscle degenerative disease patients can be determined
by measuring the Tetranor-PGDM in samples from a healthy individual
and a patient, and each measured value can then be used to
determine a "predetermined value" as a criteria for determining the
presence or absence of therapeutic efficacy according to an
ordinary method.
For example, when urine is used as a sample, the predetermined
value should preferably be determined using a daily amount of urine
pooled form each of a healthy individual and a muscle degenerative
disease patient, or urine collected at a preset time.
In the method for determining therapeutic efficacy through the
Tetranor-PGDM measurement, the concentration of the Tetranor-PGDM
contained in the urine of a patient before administration of a
therapeutic agent under controlled treatment is used as the
predetermined value, and the therapeutic agent and/or the
therapeutic method are determined as being effective when the urine
Tetranor-PGDM concentration is significantly or marginally
significantly lower than the predetermined value. The therapeutic
method and/or the administration of the therapeutic agent are then
continued. On the other hand, when there is no significant or
marginally significant decrease in the Tetranor-PGDM concentration
in urine, the therapeutic method and/or the therapeutic agent are
determined as being ineffective, and other therapeutic agents
and/or therapeutic methods are sought.
EXAMPLES
The present invention is described below in more detail based on
Example. It should be noted, however, that the invention is not
limited by the following Example.
Example 1
1. Materials and Methods
(1) Materials and Samples
The following animals were used as muscular dystrophy model
animals.
Muscular dystrophy mouse: mdx (C57Bl/10 ScSn; available from JAX
Laboratories)
Muscular dystrophy dog: CXMD.sub.J (CXMD.sub.J; available from
National Center of Neurology and Psychiatry)
For comparison, animals of the same lineage were used as
controls.
Wild-type mouse (C57BL/10 ScSn; available from JAX
Laboratories)
Normal beagle (available from National Center of Neurology and
Psychiatry)
(2) Test Compounds
The following test compounds, available as known hematopoietic
prostaglandin D synthetase (HPGDS) inhibitors, were used.
Test compound 1:
4-benzhydryloxy-1-{3-(1H-tetrazol-5-yl)-propyl}piperidine (Jpn. J.
Pharmacol., 78, 1-10 (1998))
Test compound 2:
N-methoxy-N-methyl-4-(5-benzoylbenzimidazol-2-yl-3,5-dimethylpyrrole-2-ca-
rboxamide (WO2007007778)
(3) Collection of Mouse Urine
A solvent (0.5% methylcellulose solution) or test compound 1 was
orally administered to mdx mice, 4 weeks old, for 5 days at a dose
of 30 mg/kg. Using a metabolism cage for mice, urine was collected
over the course of about 12 hours before the administration of test
compound 1 and 5 days after the administration. For comparison,
urine was also collected from wild-type mice of the same weeks old
and the same lineage used as a control. The creatinine
concentration in urine was measured using a measurement kit (L-type
Wako CRE M, Wako Pure Chemical Industries, Ltd.).
(4) Collection of Dog Urine
CXMD.sub.J was orally administered with a solvent (0.5%
methylcellulose solution) or test compound 2 for about 1 year,
followed by the administration of test compound 2 for the
solvent-administrated dog, and the solvent for the test compound
2-administered dog. Urine was collected before switching from the
solvent to test compound 2, and from test compound 2 to the
solvent. Urine was collected over time after the administered
solution was switched. For comparison, urine was also collected
from normal beagles used as a control.
(5) Urine Pretreatment
The urine (200 .mu.L) collected from the mice or dogs was mixed
with 5 ng of deuterium-labeled Tetranor-PGDM-d6 (Cayman Chemical)
used as internal standard. The volume was adjusted to 2 mL with
purified water, and pH was adjusted to 3. The urine was then
injected to a Sep-Pak Vac C18 cartridge (Waters) equilibrated with
acetonitrile (5 mL) and purified water (5 mL). The sample was
washed with a 10% acetonitrile solution (5 mL) prepared using
purified water, and with hexane (10 mL), and eluted with ethyl
acetate (5 mL) before being dried under a stream of nitrogen. The
residue was dissolved in a 10% acetonitrile solution (100 .mu.L)
prepared using purified water, and used as a measurement
sample.
(6) Tetranor-PGDM Measurement
The pretreated urine sample was used for the measurement of
Tetranor-PGDM levels. A high-performance liquid
chromatography-tandem mass spectrometry (HPLC-MS/MS) apparatus was
used for the measurement. The measurement used the HPLC apparatus
Prominence System (system controller CBM-20A, two delivery units
LC-20AD, online deaerator DGU-20A.sub.3, column oven CTO-20A,
autosampler SIL-20AC with cooling function, Shimadzu Corporation),
the guard column InertsilODS3 (inner diameter 2.1 mm.times.length
50 mm; GL Science), and the separation column InertsilODS3 (inner
diameter 2.1 mm.times.length 250 mm; GL Science). The mobile phase
had a concentration gradient of 0.01% to 0.2% formic acid or 0.01%
to 0.2% acetic acid, and acetonitrile or acetonitrile/methanol
(90:10). The flow rate was 0.2 mL/min. The column oven was set to
37.degree. C., and the autosampler to 4.degree. C. A
triple-quadrupole mass spectrometer (4000 Q TRAP LC/MS/MS system,
Applied Biosystems) that uses electrospray ionization as the ion
source was used for the MS/MS section. MRM (Multiple Reaction
Monitoring) was used for quantification. In this technique, only
the true parent ions are specifically selected from the mass of the
parent ions (precursor ion) and of the fragment ions resulting from
CID (collision-induced dissociation), and the parent ions are
accurately quantified from the area of the selected ions.
Specifically, the parent ions of the target molecule are produced
by electrospray ionization, and these parent ions are isolated by a
first mass analyzer (Q1). In a colliding section (Q2), fragment
ions characteristic of the parent ions are produced by CID
(collision-induced dissociation). The fragment ions are then
isolated in a second mass analyzer (Q3), and detected at the
detector provided downstream. Tetranor-PGDM (mass number 328) was
detected by using any of the ions with a m/z (mass number charge)
of 155, 143, and 109 produced by further decomposing the product
ions with a m/z of 327 by CID (collision-induced dissociation). The
internal standard Tetranor-PGDM-d6 (mass number 334) was detected
by using any of the product ions with a m/z (mass number/charge) of
161, 149, and 109 produced by further decomposing the product ions
with a m/z of 333 by CID (collision-induced dissociation). Data
analysis was performed with the software Analyst Version 1.4.1
attached to MS/MS. Area calculations were performed for the peaks
originating from the Tetranor-PGDM in the resulting mass
chromatogram, and each peak was quantified from the standard curve
created from the standard sample. In the quantification, correction
was made by using the area value of the peak originating from the
Tetranor-PGDM-d6 introduced as the internal standard for the
correction of the extraction efficiency and ionization efficiency
in each analysis.
(7) Symptom Evaluation
For the symptom evaluation of the mdx mice, the forefoot grip
strength was measured using a grip dynamometer for mice (traction
meter; BrainSienceldea). Each measurement was made in 2 min, and
the mean value of five trials was calculated.
2. Results
(1) Tetranor-PGDM Concentration Levels are High in the Urine of mdx
Mice
The Tetranor-PGDM concentration after correction with the urine
creatinine concentration was 17.8.+-.0.8 ng/mg Cre (mean
value.+-.standard error, p<0.0003) in the mdx mice, a value
about three times higher than the value 6.8.+-.1.0 ng/mg Cre (mean
value.+-.standard error) obtained from the wild-type mice. This
result suggests that the urine Tetranor-PGDM concentration can be
used as a urine marker for the symptom development in muscular
dystrophy.
(2) HPGDS Inhibitor Improves Symptoms in mdx Mice and Lowers Urine
Tetranor-PGDM Concentration
The effect of HPGDS inhibitor for symptoms in mdx mice was
evaluated. In contrast to the solvent-administered group that
showed no significant change in the forefoot grip strength, the mdx
mice orally administered with test compound 1 in a repeated fashion
had a significantly increased forefoot grip strength (FIG. 1,
right). The Tetranor-PGDM concentration measured in the urine of
the same mdx mice were significantly lower in the test compound
1-administered group (FIG. 1, left). The result suggests that there
is a correlation between symptom improvement and changes in urine
Tetranor-PGDM concentration in mdx mice.
(3) Tetranor-PGDM Concentration Levels are High in the Urine of
CXMD.sub.J
The muscular dystrophy model dog CXMD.sub.J had higher
Tetranor-PGDM concentration levels in urine than normal dogs, and
the Tetranor-PGDM concentration decreased in the urine of the
CXMD.sub.J administered with test compound 2 (Table 1). This result
suggests that the urine Tetranor-PGDM concentration can be used as
a urine marker for the symptom development in muscular
dystrophy.
TABLE-US-00001 TABLE 1 Tetranor-PGDM concentration in the urine of
muscular dystrophy model dogs Tetranor-PGDM concentration (ng/ml)
Normal dog (1) 5.9 Normal dog (1) 9.3 Normal dog (1) 5.0 Normal dog
(1) 5.8 CXMD.sub.J dog 27.8 HPGDS inhibitor-administered 17.9
CXMD.sub.J dog
(4) Administration of HPGDS Inhibitor Lowers Urine Tetranor-PGDM
Concentration in CXMD.sub.J
The urine Tetranor-PGDM concentration increased and the symptom
scores worsened in CXMD.sub.J that received the solvent after being
orally administered with test compound 2 for about 1 year (FIG. 2,
left). On the other hand, the urine Tetranor-PGDM concentration
decreased and the symptom scores improved in CXMD.sub.J that
received the inhibitor after being orally administered with the
solvent for about 1 year (FIG. 2, right). These results suggest
that changes in urine Tetranor-PGDM concentration can be used as a
marker for determining or predicting the effect of therapeutic
agent administration in muscular dystrophy.
* * * * *